Identification of residues/sequences in the human riboflavin transporter-2 that is important for function and cell biology

BACKGROUND: Riboflavin (RF) is essential for normal cellular metabolic activities. Human cells obtain RF from their surroundings via a carrier-mediated process that involves RF transporters -1, -2 & -3 (hRFVT -1, -2 & -3; products of SLC52A1, -A2 and -A3 genes, respectively). Little is known about the structural features of these transporters that are important for their function/cell biology. Our aim in this study was to address these issues for the hRFVT-2, a transporter linked to the neurodegenerative disorder Brown-Vialetto-Van Laere Syndrome (BVVLS). METHODS: We used comparative protein-structure modelling to predict residues that interact with two amino acids known to be critical for hRFVT-2 function (the clinical mutants L123 and L339), site-directed mutagenesis, and truncation approach in the human-derived brain U87 cell model. RESULTS: First we showed that the defect in the function of the L123 and L339 hRFVT-2 clinical mutants is related to a reduction in protein stability/translation efficiency and to retention of the protein in the ER. Mutating V120 and L121 (residues predicted to interact with L123) and L342 (a residue predicted to interact with L339) also led to a significant inhibition in hRFVT-2 function (with no change in membrane expression); this inhibition was associated with changes in protein stability/translation efficiency (in the case of V120A and L342A) and an impairment in transport function (in the case of L121). Truncating the N- and C- terminals of hRFVT-2 led to significant inhibition in RF uptake, which was associated with changes in protein stability/translation efficiency (it was also associated with a partial impairment in membrane targeting in the case of the N-terminal truncation). CONCLUSION: These investigations report on identification of residues/sequences in the hRFVT-2 protein that is important for its physiological function and cell biology.